Glucose isomerase (E.C.5.3.1.5, or xylose isomerase) is a key enzyme in the pentose phosphate pathway. It is one of the most important industrial enzymes (Kaneko et al., Bioscience, Biotechnology, and Biochemistry 2000, 64:940-947). In the food industry, it is used for the manufacture of high fructose corn syrup.
The equilibrium of the isomerization of D-glucose to fructose is primarily dictated by the temperature of the reaction. The higher the temperature, the more the fructose in the final reaction mixture. At present, the commercial glucose isomerases come mainly from Actinoplanes missouriensis, Bacillus coagulans or Streptomyces species, and are not stable at high temperature (e.g. temperature above 65° C.). Consequently, the current commercial isomerization is restricted to operate at around 60° C. and the products normally contain no more than 44% of fructose. The high fructose corn syrup containing higher fructose is therefore generated using expensive chromatographic enrichment, leading to higher production cost.
Scientists around the world have been working on the identification of thermostable and highly active glucose isomerases from thermophilic bacteria, and production of the same via protein engineering. J. G. Zeikus and his collaborators isolated and studied thermostable glucose isomerases from thermophilic bacteria, such as Thermoanaerobacterium saccharolyticum (hereinafter referred to as T. saccharolyticum) and Thermotoga neapolitana (Lee et al., Journal of General Microbiology, 139:1227-1234, 1993; Vieille et al., Methods in Enzymology, 330:215-24, 2001; Lee et al., Journal of General Microbiology, 139:1241-1243, 1993; Scriprapundh et al., Protein Engineering, 13:259-265, 2000; Scriprapundh et al., Protein Engineering, 16:683-690, 2003; Zeikus et al., U.S. Pat. No. 5,656,497). Nevertheless, the thermostabilities of the thermostable glucose isomerases from these or other bio-resources are still much to be desired, as the activities of thereof are low, and thus are not applicable to industrial applications. Therefore, glucose isomerase with high activity, or high activity and thermostability remains desirable.